Pseudomonas aeruginosa is an important cause of serious infections in humans with a predilection for lung infections in the form of either acute pneumonia in hospitalized patients (usually associated with mechanical ventilation) or chronic pneumonia in people with cystic fibrosis (CF). The central goal of the proposed project is a better understanding of the host factors contributing to pulmonary immunity to P. aeruginosa infections, with the ultimate goal of developing an intranasal vaccine for use in humans. We have shown that nasal immunization with live-attenuated aroA deletion mutants of P. aeruginosa is highly protective against pneumonia in mice caused by P. aeruginosa strains having the same lipopolysaccharide (LPS) O antigen as the vaccine (called serogroup-homologous strains). New preliminary studies have shown that intact toll-like receptor 4 (TLR4) signaling is required for optimal protective efficacy of the vaccine but not for generation of opsonic antibody while immunization of transgenic CF mice results in deficient levels of opsonic antibody yet the vaccine's protective efficacy is preserved. We recently constructed an attenuated vaccine strain that can protect against pneumonia caused by LPS-heterologous strains in the absence of serum opsonic antibody. With this vaccine, preliminary studies have found that early after bacterial challenge of wild-type mice, the cytokine IL- 17 is produced by a greater number of lung T cells, can be found at higher levels in bronchoalveolar lavage fluid compared with control vaccines, and is associated with more rapid recruitment of neutrophils to the airways. Depletion of IL-17 before challenge of immunized mice or absence of its receptor abrogates the vaccine's efficacy. These preliminary experiments have led to the hypothesis that in the adaptive immune response to P. aeruginosa in the lung, T cell-derived IL-17, and thus the recently described Th17 lineage of helper T cells, is required for optimal vaccine-induced protection. We propose to explore this hypothesis by analyzing the immunophenotypes and transcripts of T cells recruited to the lungs of immunized mice after bacterial challenge of wild-type as well as TLR4-deficient mice and transgenic CF mice. We also will determine which P. aeruginosa proteins elicit Th17 responses using a P. aeruginosa protein array. The relative contributions of Th17 cells and opsonic antibody in vaccine-induced protection from acute pneumonia and the mechanisms underlying protection will be evaluated by adoptive transfer experiments combined with passive immunization, by neutralization of IL-17 prior to challenging immunized mice, and by immunization of transgenic mice deficient in IL-17. Finally, we will assess whether expression of heterologous O antigens by the vaccine or changes in its LPS lipid A acylation pattern can enhance its effectiveness. Knowledge gained from these studies will be instrumental not only for a better understanding of the role of Th17 cells in the adaptive immune response to P. aeruginosa in the lung but also for the development of an effective vaccine against this significant human pathogen. PUBLIC HEALTH RELEVANCE: The proposed studies investigate how vaccines prevent bacterial pneumonia. In mouse models of acute pneumonia, we focus on a type of helper T cell that makes the cytokine IL-17, which works by calling in white blood cells to the lung to help fight off infection. Knowledge gained from these studies will support the development of better vaccines for pneumonia as well as an improved understanding of how to prevent lung infections in patients with cystic fibrosis.